“Phospholipase” is a general term for enzymes hydrolyzing the ester bonds in glycerophospholipid, which is a biomembrane component. Phospholipase is classified into phospholipase A1, phospholipase A2, phospholipase B, phospholipase C and phospholipase D, according to the position of hydrolysis.
Phospholipase A2 hydrolyzes the ester bond at the sn-2-position in glycerophospholipid to form fatty acid and lysophospholipid. Among the released fatty acids, arachidonic acid is metabolized into prostaglandin and leukotriene via cyclooxygenase and 5-lipoxygenase, respectively. Lysophospholipid is also metabolized into a platelet-activating factor.
That is, phospholipase A2 is considered as an enzyme initiating the formation of such lipid mediators. Inhibitors of cyclooxygenase and 5-lipoxygenase have already been used clinically as antiinflammatory drugs, and therefore, an inhibitor of phospholipase A2 located upstream of them is expected to be a potent antiinflammatory drug capable of simultaneously blocking the formation of them.
Phospholipase A2 is broadly classified into three subfamilies, i.e., secretory phospholipase A2, cytoplasmic phospholipase A2 and Ca2+ independent phospholipase A2, according to the structure and properties [J. Biol. Chem., 269, 13057 (1994)].
As to cytoplasmic phospholipase A2, three subtypes, α, β and γ, are known. Cytoplasmic phospholipase A2α, A2β and A2γ are enzymes respectively having the molecular weight of 85 kilodaltons, 110 kilodaltons and 60 kilodaltons, all of which are generally expressed in most tissues. Arginine at position 200, serine at position 228 and aspartic acid at position 549 of the amino acid sequence of cytoplasmic phospholipase A2α are essential for its activity [J. Biol. Chem., 271, 19225 (1996)] and are conserved in cytoplasmic phospholipase A2β and A2γ.
Cytoplasmic phospholipase A2α and A2β have C2 domain in the N-terminal region and Ca2+-dependently bind to phospholipid membrane via the domain. Cytoplasmic phospholipase A2γ does not have C2 domain [J. Biol. Chem., 273, 21926 (1998); J. Biol. Chem., 274, 8823 (1999); J. Biol. Chem., 274, 17063 (1999)].
Cytoplasmic phospholipase A2α is considered to participate in formation of lipid mediators by stimulus [J. Biol. Chem., 272, 16709 (1997)]. Physiological functions of cytoplasmic phospholipase A2β and A2γ have not been clarified yet.
It can be assumed that production of lipid mediators is concerned in the occurrence and progress of some diseases such as inflammation and allergy. In order to prevent or treat such diseases, there exists a need for inhibitors specific to phospholipase A2 subtype which is concerned in the diseases.
On the contrary, in view of the report that phospholipase A2 acts as a promoter of insulin secretion in pancreas [Biochimica et Biophysica Acta, 1390, 301 (1998); Biochemical Society Transactions, 25, 213S (1997); Biochemical Pharmacology, 53, 1077 (1997)], it is expected that enhancement of phospholipase A2 activity is effective for the prevention or treatment of diabetes.
In either case of inhibiting or enhancing phospholipase A2 activity, use of nonspecific chemicals is undesirable because of effect on the phospholipid metabolism in tissues and cells other than target tissues and cells.
However, the expression of cytoplasmic phospholipase A2α, β and γ is ubiquitous, and no tissue- or cell-specific cytoplasmic phospholipase A2 has so far been known.
Therefore, in order to attain the object of the present invention, it is necessary to identify and isolate phospholipase A2 concerned in specific diseases.
In the case of cytoplasmic phospholipase A2, purification and isolation from tissues or cells is not easy because it exists only in extremely small amounts. The limitation of currently employed purification methods and the difficulty in confirming that a single purified enzyme preparation has been obtained hinder the isolation of a novel subtype using conventional enzymological techniques.
Accordingly, it is expected that if a novel tissue- or cell-specific phospholipase subtype can be found and prepared in large amounts using recombinant DNA techniques, the use of such phospholipase subtype will enable the development of more specific and safer inhibitors.